Conventional chromatography is generally carried out by the passage of a sample that is to undergo chromatographic separation through a bed of spherical beads. These beads are usually packed in a tube or column in a manner to minimize interstitial volume between the beads so as to increase the efficiency of the column. The traditional synthetic route to make spherical particles is through suspension polymerization. In this kind of polymerization the choice of monomers is limited to those which are not soluble in the dispersion phase. Thus, the technique is not applicable to all polymers. This technique is easy to carry out, but the beads that are obtained are of rather polydispersed sizes. Therefore, a tedious and repetitive size fractionation must generally be carried out to obtain uniformly sized beads for packing. As a result, the packing of a column in this fashion is time-consuming and expensive.
To improve the efficiency of columns, the use of small particles or beads is desirable since such beads generally pack more easily leaving less interstitial volume. Synthesis of small porous beads is achievable, such as by a seeded polymerization, and such beads have been used in columns to achieve higher efficiencies. The use of increasingly smaller beads has, however, caused problems, since the smaller the beads, the shorter the column that is required. Certain problems associated with a short column can be solved. The column volume of such short columns is reduced (resulting in a lower separation capacity) unless reduction in length is compensated for by an increase in diameter. The best results for a separation are usually achieved when the diameter of the pores of the beads exceeds the Stockes diameter of the macromolecule to be separated by threefold or more. As a result, the beads must be very porous and therefore they are even more difficult to pack because they have a low mechanical strength. The trend to further decrease particle size in order to increase the speed and resolution of liquid chromatography columns seems to be coming to an end due to these technical limitations.
Current good quality chromatographic packings possess porosities of about 50 to 60%. Methods to increase the pore volume are known but the more porous packings that would result would likely be so brittle that their mechanical properties would not reach the standards that are expected for HPLC. Other less conventional approaches to improve column efficiency have been tried.
For example, Bio Rad manufactures Bio-Rex.RTM., 0.4 mm thick flexible disks comprising styrene-divinylbenzene ion-exchange resin (90%) in a Teflon.RTM. polymer web (10% of the overall volume) for chromatographic separation of proteins. The use of 10% Teflon, polymer, even if perfectly placed between beads, can not fully occupy the interstitial space between them, leaving some voids between particles. This will prevent the column from reaching its theoretical maximum efficiency.
PCT Publication WO 90/07965 discloses a chromatographic column plug suitable for use with gravity flow, not with pressurized flow. The plug contains cracks and channels sufficiently large to permit a hydrodynamic flow. The plug comprises a polymerized mixture of acrylic acid and methylenebisacrylamide. Hjerten et al., J. Chromatography, 473 (1989), 273-275, published 2 months after the filing of PCT Pub. WO 90/07965, discloses that the plugs disclosed in the PCT publication can not be used for chromatography, since they collapse when pressure is applied. In order to solve this problem, Hjerten recommends strongly compressing the plug to increase its resolution and ability to withstand pressure. Such compression would inherently produce non-uniform channels within the plug resulting in less than ideal column efficiency.
U.S. Pat. Nos. 4,889,632, 4,923,610 and 4,952,349 (Svec et al.) disclose thin layer macroporous membranes for so-called "membrane chromatography". The membranes are punched from a macroporous sheet of polymer and the cartridge in which they are used is different from and not a column. In fact, membrane chromatography is not chromatography since there are no repeated sorption-desorption steps as the separated molecule passes through the membrane.
Kumakura et al., J. Mat. Sci., 24 (1989), 1809-13, disclose a porous polymer composite column. The polymer material is produced from a combination of monomers by means of a radiation casting polymerization at -78.degree. C. The resulting polymer material contains extremely large holes of 10 to 40 microns in diameter, and not submicron pores. As a result, column efficiency is far less than ideal.
Accordingly, none of these approaches completely solve the problems associated with conventional packed bed chromatography columns.
EP 0,231,684 discloses chromatography columns with cast in place porous ceramic, i.e. inorganic, plugs which merely maintain separation media in place, either directly in liquid chromatography devices or indirectly as restrictors in supercritical fluid chromatographic devices. The ceramic plug is not a separation medium.
Dutch patent application No. 6,803,739 discloses the copolymerization of a solution of ethylene glycol bismethacrylate and ethylene glycol monomethacrylate in benzene in a flexible polytetrafluoroethylene tube. After polymerization, the benzene is removed and replaced by ethylene glycol monomethyl ether. The filled column that was obtained was used for gas-liquid chromatography. The column needs to be filled with ethylene glycol monomethyl ether in order to separate compounds in a gas-liquid chromatographic operation.
Thus, it is one of the objects of the present invention to produce a separation media for use in a chromatographic column that is compact and has substantially no interstitial volume.
It is another object of the present invention to produce a chromatographic column that can be prepared easily and inexpensively.
It is a further object of the present invention to produce a polymeric, i.e. organic, separation media from a large variety of monomers so that the separation media can be tailored to suit the end use of the column.
It is a further object is the production of a continuous bed which will be a separation medium for the separation of very large entities, such as protein aggregates, micelies, or nucleic acids. Certain of these large entities cannot be separated in a conventional packed column because they undergo degradation due to the shear forces in interstitial spaces between the packed particles.
These and still further objects of the present invention will be evident from the following description of the present invention.